Invertebrate choline transporter nucleic acids, polypeptides and uses thereof

Abstract

Nucleic acid encoding a choline transporter from Drosophila melanogaster , herein referred to as dmCHT, is provided. The dmCHT nucleic acids, fragments and derivatives thereof may be used to genetically alter animals, such as insects, arachnids, worms, or in cultured cells to result in dmCHT expression or mis-expression. The thus genetically altered animal or cell may find use in various screening assays to identify potential innovative pesticides or therapeutic agents by virtue of interaction with the dmCHT. The genetically altered organism or cell may also find use in studying dmCHT activity and identifying other genes that modulate the function of, or interact with, the dmCHT gene.

What is claimed is:  
     
         1 . An isolated nucleic acid or a complement thereof comprising: 
 a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2, or    a nucleotide sequence encoding a functionally active choline transporter comprising an amino acid sequence having at least 80% sequence similarity with SEQ ID NO:2,    wherein said isolated nucleic acid is less than about 15 kb.    
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid is capable of hybridizing to a nucleic acid comprising SEQ ID NO:1 using hybridization conditions comprising 6× SSC 65° C. and 0.2× SSC wash buffer at 65° C.  
     
     
         3 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid is capable of hybridizing to a nucleic acid comprising SEQ ID NO:1 using hybridization conditions comprising 5× SSC/35% formamide at 40° C. and 2× SSC wash buffer at 55° C.  
     
     
         4 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid is capable of hybridizing to a nucleic acid comprising SEQ ID NO:1 using hybridization conditions comprising 5×SSC/20% formamide at 37° C. and 1×SSC wash buffer at 37° C.  
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 220 contiguous amino acids of SEQ ID NO:2.  
     
     
         6 . The isolated nucleic acid of  claim 1 , wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 225 contiguous amino acids of SEQ ID NO:2.  
     
     
         7 . The isolated nucleic acid of  claim 1 , wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 230 contiguous amino acids of SEQ ID NO:2.  
     
     
         8 . The isolated nucleic acid of  claim 1 , wherein said nucleotide sequence encodes a polypeptide comprising the entire sequence of SEQ ID NO:2.  
     
     
         9 . A polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2.  
     
     
         10 . A polypeptide comprising an amino acid sequence having at least 80% sequence similarity with SEQ IS NO:2.  
     
     
         11 . A polypeptide comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         12 . A vector comprising an isolated nucleic acid or a complement thereof including a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2, or a nucleotide sequence encoding a functionally active choline transporter comprising an amino acid sequence having at least 80% sequence similarity with SEQ ID NO:2, 
 wherein said isolated nucleic acid is less than about 15 kb.    
     
     
         13 . A host cell comprising a vector having an isolated nucleic acid or a complement thereof including a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2, or a nucleotide sequence encoding a functionally active choline transporter comprising an amino acid sequence having at least 80% sequence similarity with SEQ ID NO:2, 
 wherein said isolated nucleic acid is less than about 15 kb.    
     
     
         14 . A method of producing a  Drosophila melanogaster  choline transporter (“dmCHT”) comprising: 
 culturing a host cell comprising a vector comprising an isolated nucleic acid or a complement thereof comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2, or a nucleotide sequence encoding a functionally active choline transporter comprising an amino acid sequence having at least 80% sequence similarity with SEQ ID NO:2, wherein said isolated nucleic acid is less than about 15 kb; and  
 recovering said dmCHT.  
 
     
     
         15 . A method of detecting at least one compound which interacts with a  Drosophila melanogaster  choline transporter (“dmCHT”) or fragment thereof, said method comprising: 
 exposing a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2 or a sequence having at least 80% sequence similarity with SEQ ID NO:2 or a fragment thereof to at least one compound; and  
 detecting at least one interaction between said at least one compound and said polypeptide.  
 
     
     
         16 . The method of  claim 15 , wherein said at least one compound comprises a putative pesticide or pharmaceutical agent.  
     
     
         17 . The method of  claim 15 , wherein exposing comprises administering said at least one compound to cultured host cells genetically altered to express said dmCHT or fragment thereof.  
     
     
         18 . The method of  claim 15 , wherein exposing comprises administering said at least one compound to an animal genetically altered to express said dmCHT or fragment thereof.  
     
     
         19 . The method of  claim 18 , wherein administering comprises at least one of bathing, feeding, injecting and contacting.  
     
     
         20 . The method of  claim 18 , wherein said compound is a putative pesticide and wherein detecting comprises observing modulations of dmCHT activity resulting in animal lethality.  
     
     
         21 . The method of  claim 18 , wherein said animal is selected from insects, arachnids and worms.  
     
     
         22 . The method of  claim 18 , wherein said animal is selected from Isopoda, Diplopoda, Chilopoda, Symphyla, Thysanura, Collembola, Orthoptera, Blattoidea, Dermaptera, Isoptera, Anoplura, Mallophaga, Thysanoptera, Heteroptera, Homoptera, Lepidoptera, Coleoptera, Spodoptera, Hymenoptera, Diptera, Siphonaptera, Arachnida, Acarinan and nematodes.  
     
     
         23 . The method of  claim 22 , wherein said animal is selected from Scistocerca spp.,  Blattella germanica, Bemisia tabaci , Myzus spp.,  Plodia interpunctella, Pectinophora gossypiella , Plutella spp., Heliothis spp., Leptinotarsa, Diabrotica spp., Anthonomus spp., Tribolium spp., Anopheles spp.,  Ctenocephalides felis, Amblyoma americanum , Meloidogyne spp. and  Heterodera glycinii.    
     
     
         24 . An animal genetically altered to express or mis-express a  Drosophila melanogaster  choline transporter (“dmCHT”), or the progeny thereof which has inherited that dmCHT expression or mis-expression mutation, 
 wherein said dmCHT comprises an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2 or a sequence having at least 80% sequence similarity with SEQ ID NO:2.  
 
     
     
         25 . The animal of  claim 24 , selected from insects, arachnids and worms.  
     
     
         26 . The animal of  claim 24 , selected from Isopoda, Diplopoda, Chilopoda, Symphyla, Thysanura, Collembola, Orthoptera, Blattoidea, Dermaptera, Isoptera, Anoplura, Mallophaga, Thysanoptera, Heteroptera, Homoptera, Lepidoptera, Coleoptera, Spodoptera, Hymenoptera, Diptera, Siphonaptera, Arachnida, Acarinan and nematodes.  
     
     
         27 . The animal of  claim 26 , selected from Scistocerca spp.,  Blattella germanica, Bemisia tabaci , Myzus spp.,  Plodia interpunctella, Pectinophora gossypiella , Plutella spp., Heliothis spp., Leptinotarsa, Diabrotica spp., Anthonomus spp., Tribolium spp., Anopheles spp.,  Ctenocephalides felis, Amblyoma americanum , Meloidogyne spp. and  Heterodera glycinii.    
     
     
         28 . A method of studying  Drosophila melanogaster  choline transporter (“dmCHT”) activity comprising: 
 detecting the phenotype caused by expression or mis-expression of dmCHT in an animal genetically altered to express or mis-express said dmCHT and/or the progeny thereof which has inherited that dmCHT expression or mis-expression mutation,  
 wherein said dmCHT comprises an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2 or a sequence having at least 80% sequence similarity with SEQ ID NO:2.  
 
     
     
         29 . The method of  claim 28  further including administering at least one compound to said animal and/or said progeny thereof and observing changes in dmCHT activity of said animal and/or said progeny thereof.  
     
     
         30 . The method of  claim 28 , wherein said animal is selected from insects, arachnids and worms.  
     
     
         31 . The method of  claim 28 , wherein said animal is selected from Isopoda, Diplopoda, Chilopoda, Symphyla, Thysanura, Collembola, Orthoptera, Blattoidea, Dermaptera, Isoptera, Anoplura, Mallophaga, Thysanoptera, Heteroptera, Homoptera, Lepidoptera, Coleoptera, Spodoptera, Hymenoptera, Diptera, Siphonaptera, Arachnida, Acarinan and nematodes.  
     
     
         32 . The method of  claim 31 , wherein said animal is selected from Scistocerca spp.,  Blattella germanica, Bemisia tabaci , Myzus spp.,  Plodia interpunctella, Pectinophora gossypiella , Plutella spp., Heliothis spp., Leptinotarsa, Diabrotica spp., Anthonomus spp., Tribolium spp., Anopheles spp.,  Ctenocephalides felis, Amblyoma americanum , Meloidogyne spp. and  Heterodera glycinii.    
     
     
         33 . The method of  claim 28 , wherein said phenotype is selected from lethality, sterility, feeding behavior, perturbations in neuromuscular function, alterations in motility, alterations in sensitivity to pesticides and pharmaceuticals, flight ability, walking, grooming, phototaxis, mating, egg-laying, responses of sensory organs, changes in the morphology, size or number of eyes, wings, legs, bristles, antennae, gut, fat body, gonads and musculature, mouth parts, cuticles, internal tissues, imaginal discs, molting, crawling, puparian formation and developmental defects in germline or embryonic tissues.  
     
     
         34 . A method of studying  Drosophila melanogaster  choline transporter (“dmCHT”) activity comprising: 
 detecting the phenotype caused by expression or mis-expression of a dmCHT in a first animal genetically altered to express or mis-express said dmCHT, and/or the progeny thereof which has inherited that dmCHT expression or mis-expression mutation, wherein said dmCHT comprises an amino acid sequence 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2 or a sequence having at least 80% sequence similarity with SEQ ID NO:2;  
 detecting the phenotype of a second animal having the same genetic mutation as said first animal and a mutation in a gene of interest; and  
 observing at least one difference between the phenotype of said first animal and the phenotype of said second animal,  
 wherein the said at least one difference identifies said gene of interest as capable of modifying the function of the gene encoding said dmCHT.  
 
     
     
         35 . The method of  claim 34 , wherein said first and said second animals are selected from insects, arachnids and worms.  
     
     
         36 . The method of  claim 34 , wherein said first and said second animals are selected from Isopoda, Diplopoda, Chilopoda, Symphyla, Thysanura, Collembola, Orthoptera, Blattoidea, Dermaptera, Isoptera, Anoplura, Mallophaga, Thysanoptera, Heteroptera, Homoptera, Lepidoptera, Coleoptera, Spodoptera, Hymenoptera, Diptera, Siphonaptera, Arachnida, Acarinan and nematodes.  
     
     
         37 . The method of  claim 35 , wherein said first and said second animals are selected from Scistocerca spp.,  Blattella germanica, Bemisia tabaci , Myzus spp.,  Plodia interpunctella, Pectinophora gossypiella , Plutella spp., Heliothis spp., Leptinotarsa, Diabrotica spp., Anthonomus spp., Tribolium spp., Anopheles spp.,  Ctenocephalides felis, Amblyoma americanum , Meloidogyne spp. and  Heterodera glycinii.    
     
     
         38 . The method of  claim 34 , wherein said phenotype is selected from lethality, sterility, feeding behavior, perturbations in neuromuscular function, alterations in motility, alterations in sensitivity to pesticides and pharmaceuticals, flight ability, walking, grooming, phototaxis, mating, egg-laying, responses of sensory organs, changes in the morphology, size or number of eyes, wings, legs, bristles, antennae, gut, fat body, gonads and musculature, mouth parts, cuticles, internal tissues, imaginal discs, molting, crawling, puparian formation and developmental defects in germline or embryonic tissues.  
     
     
         39 . A biopesticide comprising: 
 an isolated nucleic acid or a complement thereof having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% sequence similarity with 215 contiguous amino acids of SEQ ID NO:2, or    a nucleotide sequence encoding a functionally active choline transporter comprising an amino acid sequence having at least 80% sequence similarity with SEQ ID NO:2, or    a vector comprising an isolated nucleic acid or a complement thereof having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2, or a nucleotide sequence encoding a functionally active choline transporter comprising an amino acid sequence having at least 80% sequence similarity with SEQ ID NO:2,    wherein said isolated nucleic acid is less than about 15 kb.    
     
     
         40 . The biopesticide of  claim 39 , wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 220 contiguous amino acids of SEQ ID NO:2.  
     
     
         41 . The biopesticide of  claim 39 , wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 225 contiguous amino acids of SEQ ID NO:2.  
     
     
         42 . The biopesticide of  claim 39 , wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 230 contiguous amino acids of SEQ ID NO:2.  
     
     
         43 . The biopesticide of  claim 39 , wherein said nucleotide sequence encodes a polypeptide comprising SEQ ID NO:2.  
     
     
         44 . The biopesticide of  claim 39  further including at least one of a carrier and a surfactant.  
     
     
         45 . A method of controlling the growth of at least one pest, said method comprising applying to said pest and/or its locus an effective amount of a biopesticide comprising an isolated nucleic acid or a complement thereof having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% sequence similarity with 215 contiguous amino acids of SEQ ID NO:2, or a nucleotide sequence encoding a functionally active choline transporter comprising an amino acid sequence having at least 80% sequence similarity with SEQ ID NO:2, or a vector comprising an isolated nucleic acid or a complement thereof having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2, or a nucleotide sequence encoding a functionally active choline transporter comprising an amino acid sequence having at least 80% sequence similarity with SEQ ID NO:2, wherein said isolated nucleic acid is less than about 15 kb.  
     
     
         46 . The method of  claim 45 , wherein said at least one pest is selected from insects, arachnids and worms.  
     
     
         47 . The method of  claim 45 , wherein said at least one pest is selected from Isopoda, Diplopoda, Chilopoda, Symphyla, Thysanura, Collembola, Orthoptera, Blattoidea, Dermaptera, Isoptera, Anoplura, Mallophaga, Thysanoptera, Heteroptera, Homoptera, Lepidoptera, Coleoptera, Spodoptera, Hymenoptera, Diptera, Siphonaptera, Arachnida, Acarinan and nematodes.  
     
     
         48 . The method of  claim 45 , wherein said at least one pest is selected from Scistocerca spp.,  Blattella germanica, Bemisia tabaci , Myzus spp.,  Plodia interpunctella, Pectinophora gossypiella , Plutella spp., Heliothis spp., Leptinotarsa, Diabrotica spp., Anthonomus spp., Tribolium spp., Anopheles spp.,  Ctenocephalides felis, Amblyoma americanum , Meloidogyne spp. and  Heterodera glycinii.    
     
     
         49 . An antibody which specifically binds to a polypeptide comprising an amino acid sequence having 100% sequence similarity with at least 215 contiguous amino acids of SEQ ID NO:2.  
     
     
         50  An antibody with specifically binds to a polypeptide comprising an amino acid sequence having 100% sequence similarity with SEQ ID NO:2.  
     
     
         51 . A method of controlling the growth of at least one pest, said method comprising applying to said pest and/or its locus an effective amount of a biopesticide comprising an isolated nucleic acid or a complement thereof having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:2, or a vector comprising an isolated nucleic acid or a complement thereof having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:2, wherein said isolated nucleic acid is less than about 15 kb.  
     
     
         52 . The method of  claim 51 , wherein said at least one pest is selected from insects, arachnids and worms.  
     
     
         53 . The method of  claim 51 , wherein said at least one pest is selected from Isopoda, Diplopoda, Chilopoda, Symphyla, Thysanura, Collembola, Orthoptera, Blattoidea, Dermaptera, Isoptera, Anoplura, Mallophaga, Thysanoptera, Heteroptera, Homoptera, Lepidoptera, Coleoptera, Spodoptera, Hymenoptera, Diptera, Siphonaptera, Arachnida, Acarinan and nematodes.  
     
     
         54 . The method of  claim 51 , wherein said at least one pest is selected from Scistocerca spp.,  Blattella germanica, Bemisia tabaci , Myzus spp.,  Plodia interpunctella, Pectinophora gossypiella , Plutella spp., Heliothis spp., Leptinotarsa, Diabrotica spp., Anthonomus spp., Tribolium spp., Anopheles spp.,  Ctenocephalides felis, Amblyoma americanum , Meloidogyne spp. and  Heterodera glycinii .